Pro-inflammatory cytokine secretion induced by amyloid transthyretin in human cardiac fibroblasts.

Extracellular aggregates of wild-type human transthyretin are associated with heart diseases such as wild-type transthyretin (TTR)-derived amyloidosis (ATTR-wt). Due to their strategic location, cardiac fibroblasts act as sentinel cells that sense injury and activate the inflammasome. No studies of the effects of TTR amyloid aggregation on the secretion of inflammatory factors by primary human cardiac fibroblasts (hCFs) have been reported yet. The intracellular internalization of TTR aggregates, which correspond to the early stage of ATTR-wt, were determined using immunofluorescence and Western blotting of cell lysates. A further objective of this study was to analyze the secretion of inflammatory factors by hCFs after analysis of TTR amyloid aggregation using X-MAP® Luminex Assay techniques. We show that TTR aggregates are internalized in hCFs and induce the secretion of both Brain Natriuretic Peptide (BNP) and N-terminal pro B-type Natriuretic Peptide(NT-proBNP). Also, pro-inflammatory mediators such as interleukin-6 (IL-6) and IL-8 are secreted without significant changes in the levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). In conclusion, these findings suggest that IL-6 and IL-8 play important roles in the development of ATTR-wt, and indicate that IL-6 in particular could be a potentially important therapeutic target in patients with ATTR-wt.

Beneficial effects of endurance exercise training on skeletal muscle microvasculature in sickle cell disease patients.

Sickle cell disease (SCD) is a genetic hemoglobinopathy leading to 2 major clinical manifestations: severe chronic hemolytic anemia and iterative vaso-occlusive crises. SCD is also accompanied by profound muscle microvascular remodeling. The beneficial effects of endurance training on microvasculature are widely known. The aim of this study was to evaluate the effects of an endurance training program on microvasculature of skeletal muscle in SCD patients. A biopsy of the vastus lateralis muscle and submaximal incremental exercise was performed before and after the training period. Of the 40 randomized SCD patients, complete data sets from 32 patients were obtained. The training group (n = 15) followed a personalized moderate-intensity endurance training program, while the nontraining (n = 17) group maintained a normal lifestyle. Training consisted of three 40-minute cycle ergometer exercise sessions per week for 8 weeks. Histological analysis highlighted microvascular benefits in the training SCD patients compared with nontraining patients, including increases in capillary density (P = .003), number of capillaries around a fiber (P = .015), and functional exchange surface (P < .0001). Conversely, no significant between-group difference was found in the morphology of capillaries. Indexes of physical ability also improved in the training patients. The moderate-intensity endurance exercise training program improved the muscle capillary network and partly reversed the microvascular defects commonly observed in skeletal muscle of SCD patients. This trial was registered at www.clinicaltrials.gov as #NCT02571088.

Arterio-venous fistula for automated red blood cells exchange in patients with sickle cell disease: Complications and outcomes.

Erythrocytapheresis (ER) can improve outcome in patients with sickle cell disease (SCD). A good vascular access is required but frequently it can be difficult to obtain for sickle cell patients. Arterio-venous fistulas (AVFs) have been suggested for ER in SCD supported by limited evidence. We report the largest cohort of ER performed with AVFs from three French SCD reference centers. Data of SCD patients undergoing ER with AVFs in the French SCD reference center were retrospectively collected. The inclusion criteria were: SS or Sß-Thalassemia and AVF surgery for ER. SCD-related complications, transfusion history, details about AVF surgical procedure, echocardiographic data before and after AVF, AVF-related surgical and hemodynamical complications were collected. Twenty-six patients (mean age 20.5 years, mean follow-up 68 months [11-279]) were included. Twenty-three patients (88.5%) required central vascular access before AVF. Fifteen AVFs (58%) were created on the forearm and 11 (42%) on the arm. Nineteen patients (73%) had stenotic, thrombotic or infectious AVF complications. A total of 0.36 stenosis per 1,000 AVF days, 0.37 thrombosis per 1,000 AVF days and 0.078 infections per 1.000 AVF days were observed. The mean AVF lifespan was 51 months [13-218]. One patient with severe pulmonary hypertension worsened after AVF creation and died. We report the first series of SCD patients with AVF for ER, demonstrating that AVFs could be considered as a potential vascular access for ER. Patients with increased risk for hemodynamic intolerance of AVFs must be carefully identified, so that alternative vascular accesses can be considered. Am. J. Hematol. 92:136-140, 2017. © 2016 Wiley Periodicals, Inc.

Extracellular Calpain/Calpastatin Balance Is Involved in the Progression of Pulmonary Hypertension.

Excessive growth of pulmonary arterial (PA) smooth muscle cells (SMCs) is a major component of PA hypertension (PAH). The calcium-activated neutral cysteine proteases calpains 1 and 2, expressed by PASMCs, contribute to PH but are tightly controlled by a single specific inhibitor, calpastatin. Our objective was to investigate calpastatin during pulmonary hypertension (PH) progression and its potential role as an intracellular and/or extracellular effector. We assessed calpains and calpastatin in patients with idiopathic PAH and mice with hypoxic or spontaneous (SM22-5HTT(+) strain) PH. To assess intracellular and extracellular roles for calpastatin, we studied effects of the calpain inhibitor PD150606 on hypoxic PH in mice with calpastatin overexpression driven by the cytomegalovirus promoter (CMV-Cast) or C-reactive protein (CRP) promoter (CRP-Cast), inducing increased calpastatin production ubiquitously and in the liver, respectively. Chronically hypoxic and SM22-5HTT(+) mice exhibited increased lung calpastatin and calpain 1 and 2 protein levels and activity, both intracellularly and extracellularly. Prominent calpastatin and calpain immunostaining was found in PASMCs of remodeled vessels in mice and patients with PAH, who also exhibited increased plasma calpastatin levels. CMV-Cast and CRP-Cast mice showed similarly decreased PH severity compared with wild-type mice, with no additional effect of PD150606 treatment. In cultured PASMCs from wild-type and CMV-Cast mice, exogenous calpastatin decreased cell proliferation and migration with similar potency as PD150606 and suppressed fibronectin-induced potentiation. These results indicate that calpastatin limits PH severity via extracellular mechanisms. They suggest a new approach to the development of treatments for PH.

Calpastatin overexpression impairs postinfarct scar healing in mice by compromising reparative immune cell recruitment and activation.

The activation of the calpain system is involved in the repair process following myocardial infarction (MI). However, the impact of the inhibition of calpain by calpastatin, its natural inhibitor, on scar healing and left ventricular (LV) remodeling is elusive. Male mice ubiquitously overexpressing calpastatin (TG) and wild-type (WT) controls were subjected to an anterior coronary artery ligation. Mortality at 6 wk was higher in TG mice (24% in WT vs. 44% in TG, P < 0.05) driven by a significantly higher incidence of cardiac rupture during the first week post-MI, despite comparable infarct size and LV dysfunction and dilatation. Calpain activation post-MI was blunted in TG myocardium. In TG mice, inflammatory cell infiltration and activation were reduced in the infarct zone (IZ), particularly affecting M2 macrophages and CD4(+) T cells, which are crucial for scar healing. To elucidate the role of calpastatin overexpression in macrophages, we stimulated peritoneal macrophages obtained from TG and WT mice in vitro with IL-4, yielding an abrogated M2 polarization in TG but not in WT cells. Lymphopenic Rag1(-/-) mice receiving TG splenocytes before MI demonstrated decreased T-cell recruitment and M2 macrophage activation in the IZ day 5 after MI compared with those receiving WT splenocytes. Calpastatin overexpression prevented the activation of the calpain system after MI. It also impaired scar healing, promoted LV rupture, and increased mortality. Defective scar formation was associated with blunted CD4(+) T-cell and M2-macrophage recruitment.

Reconciling depressed Ca2+ sparks occurrence with enhanced RyR2 activity in failing mice cardiomyocytes.

Abnormalities in cardiomyocyte Ca2+ handling contribute to impaired contractile function in heart failure (HF). Experiments on single ryanodine receptors (RyRs) incorporated into lipid bilayers have indicated that RyRs from failing hearts are more active than those from healthy hearts. Here, we analyzed spontaneous Ca2+ sparks (brief, localized increased in [Ca2+]i) to evaluate RyR cluster activity in situ in a mouse post-myocardial infarction (PMI) model of HF. The cardiac ejection fraction of PMI mice was reduced to ∼30% of that of sham-operated (sham) mice, and their cardiomyocytes were hypertrophied. The [Ca2+]i transient amplitude and sarcoplasmic reticulum (SR) Ca2+ load were decreased in intact PMI cardiomyocytes compared with those from sham mice, and spontaneous Ca2+ sparks were less frequent, whereas the fractional release and the frequency of Ca2+ waves were both increased, suggesting higher RyR activity. In permeabilized cardiomyocytes, in which the internal solution can be controlled, Ca2+ sparks were more frequent in PMI cells (under conditions of similar SR Ca2+ load), confirming the enhanced RyR activity. However, in intact cells from PMI mice, the Ca2+ sparks frequency normalized by the SR Ca2+ load in that cell were reduced compared with those in sham mice, indicating that the cytosolic environment in intact cells contributes to the decrease in Ca2+ spark frequency. Indeed, using an internal "failing solution" with less ATP (as found in HF), we observed a dramatic decrease in Ca2+ spark frequency in permeabilized PMI and sham myocytes. In conclusion, our data show that, even if isolated RyR channels show more activity in HF, concomitant alterations in intracellular media composition and SR Ca2+ load may mask these effects at the Ca2+ spark level in intact cells. Nonetheless, in this scenario, the probability of arrhythmogenic Ca2+ waves is enhanced, and they play a potential role in the increase in arrhythmia events in HF patients.

Sleep-disordered breathing in chronic heart failure: development and validation of a clinical screening score.

AIMS: Sleep-disordered breathing (SDB) is highly prevalent and of adverse prognostic significance in patients with chronic heart failure (CHF). Polygraphy is used for diagnosing SDB but polygraphy resources fall short of needs. Here, our aim was to develop a score for SDB screening in patients with CHF. METHODS AND RESULTS: Consecutive patients with stable chronic CHF referred to our CHF clinic for a scheduled follow-up evaluation were included prospectively between 2000 and 2012. SDB was defined as an apnoea-hypopnoea index ≥ 5/h as assessed by routine polygraphy. A screening score was developed as a linear combination of factors independently associated with SDB by multivariate logistic regression. Calibration and discrimination were evaluated using the Hosmer-Lemeshow (HL) test and area under the receiver-operating characteristics curve (AUC), respectively. Bootstrapping was performed to assess internal validity. Of 450 included patients (mean age, 59.5 ± 13.7 years), 397 (88%) had SDB. An easy-to-use score was based on age (2 points if ≥65 years), body mass index (2 points if ≥25 kg/m(2)), New York Heart Association (NYHA) class (2 points if ≥3 or 4) and male gender (3 points). A score cut-off of 5 was 78.9% sensitive and 61.5% specific for SDB. The final model exhibited adequate calibration (pHL ≥ 0.3) and discrimination (AUC, 0.737; 95% confidence interval, 0.663; 0.810). CONCLUSION: An easy-to-use clinical score combining age, body mass index, NYHA class, and gender may help to identify those CHF patients most likely to have SDB, thereby improving the allocation of scarce polygraphy resources and early diagnosis of SDB.

Changes in three-dimensional speckle-tracking-derived myocardial strain during percutaneous coronary intervention.

BACKGROUND: The aim of this study was to evaluate changes in three-dimensional (3D) speckle-tracking-derived myocardial strain during ischemia. METHODS: Twenty patients referred for percutaneous coronary intervention were studied (mean age, 65 ± 11 years; mean left ventricular ejection fraction, 56 ± 7%). Of the 20 study patients, 12 had severe left anterior descending coronary artery stenosis. A full-volume apical view using multibeat 3D modality and two-dimensional (2D) apical views (four chamber, two chamber, and three chamber) were recorded during coronary occlusion before wall motion abnormalities. After percutaneous coronary intervention, ultrasound contrast agent was selectively delivered through the target lesion to delineate ischemic and peri-ischemic segments (ischemia-adjacent segments). Strain values derived from 2D and 3D speckle-tracking echocardiography were compared in ischemic and nonischemic segments. RESULTS: Despite no changes in wall motion and 2D left ventricular ejection fraction (56 ± 7% vs 56 ± 7%), global longitudinal strain by 2D imaging was impaired during percutaneous coronary intervention (-16 ± 3% vs -14 ± 3%, P = .01). Similar changes were observed for all 3D strain components: -11 ± 16% for longitudinal (-15 ± 4% vs -13 ± 4%, P = .03), -13 ± 25% for circumferential (-15 ± 4% vs -12 ± 4%, P = .02), -12 ± 16% for area (-25 ± 5% vs -22 ± 6%, P = .009), and -12 ± 25% for radial global strain (39 ± 12% vs 33 ± 12%, P = .04). During coronary occlusion, 2D longitudinal peak strain and all 3D peak strain components decreased in ischemic segments, while no changes was observed in nonischemic segments. In peri-ischemic segments, only 3D longitudinal and area strain were impaired during ischemia. CONCLUSIONS: Changes in myocardial deformation related to ischemia can be characterized by 3D speckle-tracking echocardiography before the stage of wall motion abnormality.

Left ventricular SERCA2a gene down-regulation does not parallel ANP gene up-regulation during post-MI remodelling in rats.

BACKGROUND: In most animal models of chronic hemodynamic overload of the left ventricle (LV) as well as in human end stage heart failure, the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) mRNA levels are decreased in parallel with increased atrial natriuretic peptide (ANP) mRNA levels. The situation in the remote myocardium following myocardial infarction (MI) is unclear. AIMS: (1) To examine SERCA2a mRNA levels in the non-infarcted LV myocardium of rats at the chronic stage of experimental MI and (2) To examine whether a negative linear correlation exists between SERCA2a and ANP mRNA levels in this model. METHODS: Anesthetized adult male Wistar rats underwent left coronary artery ligation or sham operation. Three months later, the rats were divided into three groups: sham-operated rats (sham, n=21), HF-free rats with MI (non-failing (NF)-MI, n=29) and rats with both MI and HF (congestive heart failure (CHF)-MI, n=14). LV remodelling and function were assessed by echocardiography and hemodynamic measurements. SERCA2a and ANP mRNA levels were determined by Northern and dot blot analysis with specific cDNA probes. RESULTS: LV SERCA2a mRNA levels varied markedly in sham-operated rats (0.9-1.8). Mean ANP mRNA level increased markedly and mean SERCA2a mRNA level decreased moderately in the remote myocardium. In some NF-MI rats, SERCA2a mRNA levels were higher than those in some sham controls. Whereas ANP mRNA levels correlated well with MI severity (r2=0.79, p<0.001), this was not the case for SERCA2a mRNA levels (r2=0.42, p<0.01). We found no negative correlation between ANP and SERCA2a mRNA levels. CONCLUSION: SERCA2a gene down-regulation in the non-infarcted myocardium of rats with MI does not correlate with ANP gene up-regulation, suggesting that the two genes are not antithetically regulated.

Constitutive cardiac overexpression of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase delays myocardial failure after myocardial infarction in rats at a cost of increased acute arrhythmias.

BACKGROUND: Heart failure often complicates myocardial infarction (MI), and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA2a) is underexpressed in the failing myocardium. We examined the effect of preexisting cardiac SERCA2a protein overexpression on rat survival and left ventricular (LV) remodeling after MI. METHODS AND RESULTS: Baseline myocardial SERCA2a expression was 37% higher in transgenic (TG) rats than in their wild-type (WT) controls, consistent with enhanced myocardial function. The mortality rate of TG rats during the 24 hours after surgical MI was higher than that of WT rats (71% versus 35%, P<0.001), associated with a higher frequency of ventricular arrhythmias, and was normalized by lidocaine treatment. The increased acute-phase mortality in TG rats was not accompanied by increased 6-month mortality. Function of the noninfarcted myocardium, as assessed by tissue Doppler imaging, was higher in TG rats than in WT rats for up to 1 month after MI, a beneficial effect no longer observed at 3 months. LV remodeling and global function were similar in TG and WT rats. No difference in papillary muscle function was found at 6 months. CONCLUSIONS: Constitutive cardiac SERCA2a overexpression has a transient beneficial effect on remote myocardium function in rat MI, with no improvement in LV global function or prevention of LV remodeling and failure. This benefit is associated with a higher risk of acute mortality, which is prevented by lidocaine treatment.